Antibiotic-containing animal feed



United States Patent ice 3,304,227

Patented Feb. 14, 1967 3,304,227 T e accepted nomenclature regards thetetracycline ANTIBIOTIC-CONTAINING ANIMAL FEED (achromycm) as Loyal E.Loveless, 248 Rosemont, St. Louis, Mo. 63119 H3O OH CH3 CH1 No Drawing.Continuation of application Ser. No.

218,553, Aug. 22, 1962. This application July 15, H f 1965, Ser.No.472,361 H/ H H 4 Claims. (Cl. 167-531) H OH The present application is acontinuation of copending H i CNH application Serial No. 218,553, filedAugust 22, 1962, y l i H now abandoned. H 1 This invention relates totetracyclic antibiotics and to OH 0 OH O procedures for using themeffectively. More specifically, 0H (2) the invention is directed to theuse of tetracycline anti- The substituted tetracyclines include: bioticsin the control of bacteria and other pathogenic Substiment Amine morganisms in the bodies of higher animals, particularly (Terramycin)sdoxytetracycline birds and mammals. More specifically, the inventionre- (Aureomycin)7 chlol otetracyciine lates to animal feeds containingtetracycline antibiotics lbromotetrawchm in an effective formulation.

When a living organism ingests a tetracycline antibiotic, otherhomologues include? especially tetracycline, S-oxytetracycline or7-chlorotetra- 2O cycline, it enters the body fluids and becomes activein the control of parasitic bacteria and other pathogens present in thesaid fluid. Often the control of these organisms depends upon asubstantial concentration of the antibiotic in the body fluid,especially in the blood. In some instances, the effective concentrationcan be attained by increasing the quantity charged to the feed, but thisprocedure is often not desirable because of the cost of .the

6-deoxy-S-oxytetracycline 4-de dimethylamino -5-oxytetracycline 6ieoxytetracycline 6-deoxy-6-dimethyltetracycline 6-methyl-7-chlorotetracycline 4-de (dimethylamino) tetracycline 4-de dimethylamino-7-chlorotetracyc1ine 6-demethyltetracycline tetracycline antibiotic andbecause of the inefficient utilizaother tetracyclines the basicStructure are:

tion of the additional amount. By analytical procedures 4 di h 1 i 1 4,45,7,8,9,10-decanhydr0-3,11,1221- the concentration in the blood can bedetermined and h h 10- i g h h this is a measure of the degree ofprotection which the boxamide host animal is receiving from theantibiotic. If the carboxamidmN-(isopropyl) 6 dcoxy 7chl,o,rotetmcycline animal is seriously infected the use of theantibiotics may 6 demethy1 12a-deoxyanhydrotetracycline be manifested bya growth response or an improvement 30 5a epitetracyc1ine feed F FY-6-methylene-5-oxytetracycline The availability of the tetracyclineantibiotics and the 6 demethy1 7 ch1orotetmcyc1ine concentration in theblood may under certain conditions 7 bmm0 6 dimethyp@deoXYtetracYcline 9influenced by other food K P P 50111121? s12a-dibromo-l,4,4a,5,12,12a-hexahydro-3,10,1l-trihycium compoundsinhibit the assimilation of the antibiotics droxy 6 methyi l,12 di0X0 2naphthacenecarboxamide and it is desirable to keep them at a minimum byeither reducing the calcium content or by substituting veryinotherfeompehhds eehtaihihg the basic configuration (1) solublecompounds, such as calcium sulfate, for the more and h watehselhhle h falso embraced by the soluble calcium carbonate and limestone usuallyused. term tetmeyehhe ahhblehes' The use an of these The inhibitingeffect of calcium can also be minimized Compounds in the eohtrohofparasitic e e and other by the addition of sodium salts whereby theobjectionable pathogens and esheelahy h the y hulds 1s Weh khewh calciumis replaced by sodium and potassium Thus and not part of this invention.It is also known that the sodium Phosphate (Nspo) dibasic sodiumphosphate tetracycline antibiotics enter the bloodstream and that the(NBQHPOQ and dihydric phosphate (NaHzpO) may concentration of theantibiotics in the blood is a measure added of the ability of the animalto resist the deleterious effects The phrase tetracycline antibiotics asused in this the m i known by h specification and the appended claims isintended to have h the qhehhty of the h e Introduced m the digesageneric significance and includes avariety of compounds h tract theConcentrahon m the bloodstream can be which have antimicrobial activityand have similar struc- Increased tures to which have been assigned thegeneric name tet- It been fohhd that by mmWWIFFmFmdJCtmH racycline bythe Chemical Abstracts Nomenclature of certa n chemcials greatlyincreased antibiotic concen- (S.A.C.S. 74, 4976). Many of thetetracycline antibiotics trahehs m h bieed 9 Although the phenom areproduced by culturing certain fungi and separating the enoh ahhbloncpotenhahon is known eompouhds bacterial active compounds produced by thefungi. Other used hh manner not greatly pr tetracycline antibiotics aresynthesized by the chemical only mlhhhal behehelal f The primary Purposemodification of naturally produced tetracycline antibiotics. i Pmvld?eomposlhehs Whleh h fed 9 i i i W111 The tetracycline antibiotics willhave the basic structure: Induce hlgh levels of the tetracyclineanhblohes m the bloodstream. A further purpose is to provide a procedure& which will enable the animals to become more resistant to 5 harmfulbacteria and will provide for a more eflicient use of the antibiotic. 0HIt is known that terephthalic acid when ingested with the tetracyclineantibiotics aids in the assimilation of the ONHQ antibiotic, such thatthe concentration of the antibiotic I g 7 in the blood is substantiallyincreased. Much experimen- OH (1) tal work has been done and manyobservations have been with siibstituents which may vary in position andkind. made with respect to the desirable antibiotic potentiation effectof terephthalic acid. Compounds with equivalent antibiotic potentiationactivity will have unquestionable utility. In the experimental workdescribed hereinafter the activity of potentiating compounds are inseveral instances described in the percentage of the activity ofterephthalic acid.

In accordance with this invention it has been found that aromaticpolycarboxylic acids including terephthalic,

acid are of exceptional activity as potentiatin-g agents if they areused in the presence of surface active agents. The mechanism of theactivation has not been definitely ascertained, but it is of great valuein providing for a more economical utilization of tetracyclineantibiotics.

Example 1 In the absence of surface active agents the following datawere observed in feeding chicks in the one day exclusion test. Three tofour week old chicks were raised in starting batteries on anantibiotic-freediet. On the test day the chicks were weighedindividually and each given 75 mg. of S-oxytetracycline per kg. of bodyweight and 400 mg. of the test compound per kg. of body weight. Threehours after the administration of the antibiotic, blood was withdrawn byheart puncture, centrifuged and the plasma assayed for the antibioticconcentration. The fol-lowing data sets forth the percentage ofterephthalic acid activity observed by the several aromaticpolycarboxylic acids:

Terephthalic acid (stem closed) 100 Phthalic acid 1751,2,3-tricarboxybenzene 210 1,2,4-tricarboxybenzene 1401,3,5-tricarboxybenzene 155 l,2,4,S-tetracarboxybenzene 225 Thisexperiment demonstrates the utility of the class of aromatic carboxylicacids in the same manner as the prior art compound terephthalic acid.

The surfactants which can be used in the practice of this invention maybe any of a large number of compounds which have both hydrophobic andhydrophilic properties. A wide variation of molecular configurations andchemical properties are possible. These surface active compounds mayhave cationic or anionic properties or may be neither cationic noranionic (nonionic). Some surface active agents may have both cationicand anionic functional configurations, which agents are known asampholytic surfactants.

Several distinct types of anionic surfactants may be used, for examplethe straight chained naturally occurring fatty acids and their soaps.This type includes the fatty acids derived from animal and vegetableoils by the conventional saponification procedures, such as stearicacid, ricinoleic acid, mar-garic acid, lauric acid, myristic acid,palmitic acid, capric acid, caprylic acid, oleic acid, linoleic acid andlinolenic acid, the alkali metal salts of these acids, particularly thesoduim and postassium salts, the heavy metal soaps of these acids, suchas the salts of rnetals such as lead, cobalt, manganese, Zn, nickel,aluminum, copper, iron and chromium, and the amino soaps, wherein thesaid acids are reacted with amines, for example as indiethylaminostearate, di-n-butylaminolaurate and anilinopalmitate.

Modified carboxylic acids of somewhat different properties may be madeby substituting organic groups on the aliphatic chain, for example byoxidation, which can take place on an unsatrated linkage to form ahydroxyl or epoxy group. Formic acid may be added to a double bond. Byhalogenation of the alpha carbon atom, hydroxylated alkyl substituentscan be introduced. Halogenation on the unsaturated bonds provides ameans of introducing side chains or functional groups. Other groups canbe added directly to the double bond, for example formaldehyde willreact with oleic acid to form a six member oxygen heterocycli'csubstituent. A ya pounds.

riety of other mixed carboxylic acids can be prepared by saponificationof wool, wax or by separation from tall oil. ther acids can be preparedfrom paraflins by oxidation to carboxy, keto and hydroxyl groupsaccompanied by the formation of lower molecular weight corn- Thesecarboxylic acids may have intermediate groups such as ester, ether,sulfonyl. All of these modified carboxylic acids may be used as surfaceactvie agents or may be converted to salts of alkali or heavy metals, orto amino soaps.

Another group of anionic surfactants are the esters of sulfur acids,such as sodium lauryl sulfate, the various alkali metal alkylarylsulfonates, for example sodium dodecylbenzene sulfonate,sodium-Z-ethylhexylnaphthyl sulfonate and sodium octadecyl benzenesulfonate. These and other sulfuric acid esters can be prepared by firstreducing the acids, for example by sodium or by hydrogenation followedby sulfonation. Oxo alcohols and other synthetic alcohols, such as kerylalcohols, made by chlorination of kerosene, reacting with sodiumbenzoate and saponifying the resulting product; or the alcohol made byreaction of formaldehyde with triisobutylene may also be sulfated.Olefins or olefin polymers (polypropylenes and isobutylene polymers) maybe sulfated directly. Ester alcohols and amidoalcohols will bysulfonation procedures provide valuable surfactants. Many alkanesulfonates, for example octadecyl sulfonate, those prepared by oxidationof long chain alkyl mercaptans, those prepared by direct reaction ofparaffin oils with S0 or chlorosulfonic acid are valuable anionicsurfactants. Petroleum sulfonates, known as mahogany or green soaps,by-products from the petroleum refining industry, are useful.

Other types of anionic surfactants are the phosphorus compounds such asdi(2-ethylhexyl)orthophosphate or the acid phosphate esters ofcoco-monoethanolamide; sulfinic acid made by reduction of sulfonylchlorides, the sulfunamides, the hydrogenated aromatization orpolymerization of tall oil rosin acids, the lignin sulfonate by-productsfrom the sulfite paper industry and the lignin sulfonates modified bycontrolled alkaline hydrolysis. These anionic surfactants have a longchain oil soluble group and an ionizable acid or salt group to providethe anionic properties.

The useful surfactants for the practice of this invention also includemany of the cationic types; for example, the fatty acid nitrilesprepared by the reaction of ammonia and fatty acid under pressure atelevated temperatures, amines with or without intermediate esters, etheror amide linkages; the amino alcohols; allyl d-iamines; the alkylanilines; the imidazolines made by condensing fatty acids with ethylenediamine; the quaternary nitrogen bases containing a long chain oilsoluble group and an ionizable acid group, usually chloride or bromide,such as tetradecyl pyridinium chloride; N-alkyl morpholine, which may bequaternized, for example with butyl bromide; the reaction product ofpyridine and Z-chloroethyl dimethyl dodecyl ammonium chloride; thereaction products of tertiary amines and chloromethyl stearamide, thethiouronium salts, such as the reaction product of chlorinated paraffinand urea; and the phosphonium salts, such as higher alkyl bis(dimethylamino phosphonium halides.

Another and a very important type of surfactant, which can be used, isthat having neither anionic or cationic properties. These are callednonionic surfactants and include esters, ethers, alcohols and phenolswhich do not have ionizable substituents. They must have an oilattractive long chain hydrocarbon group and a hydrophilic substituentwhich is nonionogenic. This type of surfactant includes polyhydroxycompounds such as the fatty acid esters of the polysaccharides, such assucrose and dextrose; the fatty acid monoesters of glycols; esters offatty acid monoglycerides and hydroxy acids, such as lactic or glycolicacids; and the glucose derivatives, for example the transesterificationproduct of methyl glucoside and long chain fatty acid esters. The bulkof the nonionic surfactants are based on ethylene oxide or homologuesthereof, wherein the hydrophilic properties are due to the presence of aplurality of ethoxy groups. Many of these are prepared by thecondensation of a large excess of ethylene oxide on a nonionichydrophobic phenol or alcohol, for example dodecylphenol, but they alsocan be prepared by reacting a long chain fatty acid with a large excessof ethylene oxide which reacts with the ionogenic carboxy groups to formhydroxy ethyl esters, for example ethoxy (ethoxy) stearate, and theethylene oxide (9 mol) condensate with tetrapropylene-benzene sulfonicacid. An increase in the number of mols of ethylene oxide increases thehydrophilic properties. The nonionic surfactants may have in thehydrocarbon structure non-hydrocarbon groups such as tertiary aminonitrogen, sulfide, sulfone and ester groups. The intermediates forcondensation with ethylene oxide have terminal groups such as hydroxyl,aldehyde, carboxyl, mercapto, sulfonic acid, sulfonamide, guanylurea,amino and amido. These will provide the necessary surface activeproperties.

One additional type of surfactants are the ampholytic compounds,possessing both cationic and anionic groups, for exampleN-dodecyl-N-phenyl, amino carboxylic acids, or salts, dimethylaminoethylbenzene sulfonic acid quaternized with an alkyl halide and thecarboxylic acids containing an imidazoline molecular grouping.

Frequently the surface active agents may be improved or otherwisemodified by the incorporation of additives known as builders such aspyrophosphates, polyphosphates, sodium silicates, clays, particularlythe expanding lattice type silica gels, sodium Zincate, colloidalaluminum hydroxide, ammonium carbonate, dicyandiamide, and sodiumferrocyanide.

Other surfactants and methods for their preparation and use aredescribed in detail in the textbook Surface Active Agents andDetergents, Schwartz, Perry and Berch, Interscience Publishers Inc.(1958). The word surfactants is used to define the broad class, all ofwhich appear to aid the absorption of the tetracycline antibiotics andtransfer from the digestive system into the bloodstream.

The following surfactants were used in the antibiotic potentiationstudies described hereinafter. In the following tables the surfactantsare identified by the letter associated with each of the followingdescribed surfactants:

(A) dodecylbenzene sodium sulfonate (85 active) (B) dodecylbenzenesodium sulfonate (70% active) (C) dodecylbenzene sodium sulfonate (40%active) (D) mixed acids derived from tallow condensed with ethyleneoxide (E) alcohols from tallow acids condensed with ethylene oxide (F)long chain mixed mercaptans condensed with ethylene oxide (G) dodecylphenol condensed with 6 mols of ethylene oxide (H) nonyl phenolcondensed with ethylene oxide (1) long chain alcohols condensed withethylene oxide (85% active) (I n-mixed alkyl, 8-18 carbon atoms,trimethyl ammoniurn chloride (K) 1,3-propane diamine particularlyquaternized with halides derived from tallow oil and condensed withethylene oxide Example 2 In vivo potentiati-on studies were conductedwith 3 to 4 week old chicks maintained on an antibiotic free diet for atleast one week. The tests were conducted by weighing each birdindividually and administering to each bird 75 mg. of 5-oxytetracyclineand 400 mg. of one of the above surfactants per kilogram of body weightby intu-bation. As a control, birds were fed the same amount of5-oxytetracycline with no surfactant. Samples of blood 6 were takenthree hours after treatment with antibiotic. The samples werecentrifuged to obtain a clear plasma and the plasma assayed for contentof 5-oxytetracycline. The following table sets forth the observedconcentrations of the antibiotic as a percentage of that found in thebirds fed the antibiotic Without surfactants:

Percent of control birds on normal ration These experiments demonstratethat surface active agents provide a very substantial increase, from 160to 455 percent, in the concentration of antibiotic in the bloodstream inanimals.

Example 3 Two to four week old birds were divided into groups of 15chicks, which were weighed as a group and fed with each of the followingdiets:

Ingredient Diet A Diet B Ground yellow corn 60. 2625 60. 2625 50%soybean oil meal 24. 0 24. 0 Tallow 2 2. 5 2. 5 Fish meal 5. 0 5. 0Dehydrated alfalfa meal I 1. 0 1. 0 Corn gluten meal (43%) 2. 0 2. 0Vitamins O. 5 0. 5 I-Iydroxy analogue of methionine 0. 0375 O. 0375Terrarnycin 0. 2 0. 2 Potentiator A 0. 375 O. 50 Cellulose filler 0. 3750.250 Mineral mixture 3. 75 3. 75

After one week on the above diets 2 ml. of blood were taken from theheart of each bird and the blood samples for each group of birdscombined, centrifuged and assayed for 5-oxytetracycline. The blood ofthe control birds fed the same diet without the potentiators was foundto contain 7.05 micrograms per ml. of the antibiotic. The blood of birdsfed feeds containing 0.375 percent of potentiator A (dodecylbenzenesodium sulfonate-% active) was found to contain 150 percent of theantibiotic of the control. The blood of birds fed on feeds with 0.50percent of Potentiator A was found to contain 215% of the controlconcentration of the antibiotic. The blood of birds fed 0.5 percent ofthe dodecylbenzene sodium sulfonate (85% active) and 0.5 percent ofterepht-halic acid was found to contain 285 percent of the concentrationof the blood in the birds fed without potentiators.

Example 4 Using the procedure of Example 2 except that the bird were fedthe 5-oxytetracycline with 400 mg. per kg. body weight of terephthalicacid and 400 mg./kg. of surface active agents identified above bychemical name and reference letter.

Surfactant: Percent control (A) 620 (D) 305 (E) 470 Example 5 Theprocedure of Example 4 was repeated except that 7-chlorotetracycline wasused in place of 5-oxytetracycline. It was found that surfactant A incombination with terephthalic acid increased the concentration in theblood serum percent.

This invention may be practiced by preparing animal feeds byincorporating tetracycline antibiotics, surfactants and aromaticpolycarboxylic acids in a conventional mixture of feed components. Theuse of this combination of potentiators produces a greater potentiati-oneffect than is attained from either component alone. The same beneficialeffect can be obtained by adding the surfactant to feed compositionscontaining terephthalic acid and tetracyclic antibiotics or by addingboth surfactants and aromatic polycarboxylic acids to conventional feedswhich usually include tetracycline antibiotics.

In' the preparation of feeds the tertacycline antibiotics may be presentin the feed in the amount of 50 to 500 gins. per ton of feed, preferredusage being from 150 to 300 gms. per ton. The potentiators have abroader range of activity for example one to twenty-five pounds per tonof feed, being the same for both the aromatic polycarboxylic acids andfor the surface active agents. The optimum ratio of the two potentiatingagents will depend upon the relative activity and the cost of each. Thequantity of the potentiating mixture is usually much greater than thatof the tetracycline antibiotics and will be determined byexperimentation to ascertain what will provide the most efficientutilization of the antibiotic. Most compositions will find the optimumproportion between and pounds per ton of feed for both thepolycarboxylic aro matic acids and the surface active agents.

Although the invention is described with respect to specificmodifications, it is not intended that the details thereof shall belimitations on the scope of the invention except to the extentincorporated in the following claims.

What is claimed is:

1. A method of augmenting animal blood concentration of antibioticsselected from the class consisting of 5-oxytetracycline and7-chlorotetracyoline which consists essentially in feeding birds about75 mg. per kg. body weight of an antibiotic selected from the classconsisting of S-oxytet-racycline and 7-chlorotetracycline in combinationwith about 400 mg. per kg. body weight of a benzene polycarboxylic acidof the class consisting of terepthalic acid, .phthalic acid,1,2,3-tricarboxybenzene, 1,2,4-tricarboxybenzene,1,3,5-tricarboxybenzene, and l,2,4,5-tetracarboxybenzene, and about 400mg. per kg. body weight of surface active agents selected from the classconsisting of dodecylbenzene sodium sulfonate, mixed acids derived fromtallow condensed with ethylene oxide and mixed alcohols derived fromtallow condensed with ethylene oxide.

2. A method of augmenting animal blood concentra tion of antibioticsselected from the class consisting of S-oxytetracycli-ne and'7-chlorotetracycline which consists essentially in feeding birds acomposition containing from about 50 to about 500 grams per ton of feedof antibiotics of said class, about one to about 2.5 pounds per ton offeed of a benzene polycarboxylic acid selected from the class consistingof terephthalic acid, phthalic acid, 1,2, 3-tricarboxybenzene,1,2,4-tricarboxybenzene, 1,3,5-tricarboxybenzene and1,2,4,S-tetracarboxybenzene, and from about one to about 25 pounds perton of feed of a surface active agent of the class consisting ofdodecylbenzene sodium sulfonate, mixed acids derived from tallowcondensed with ethylene oxide, and mixed alcohols derived from tallowcondensed with ethylene oxide.

3. An animal feed containing from about 50 to about 500 grams per ton offeed of an antibiotic selected from the class consisting of5-oxytetracycline and 7-chlorotetracycline, about one to about 25 poundsper ton of feed of a benzene polycarboxylic acid selected from the classconsisting of terephthalic acid, phthalic acid, 1,2,3-tricarboxybenzene,1,2,4-trica-rboxybenzene, 1,3,5-tricarboxybenzene and1,2,4,S-tetracarboxybenzene, and from about one to about 25 pounds perton of feed of a surface active agent of the class consisting ofdodecylbenzene sodium sulfonate, mixed acids derived from tallowcondensed with ethylene oxide, and mixed alcohols derived from tallowcondensed with ethylene oxide.

4. A composition for augmenting blood levels in host animals ofantibiotics selected from the group consisting of S-Qxytetracycline and7-chlorotetracycline which cornprises from about one to about 10 partsby weight of said antibiotic, from about 10 to about 250 parts by weightof a benzene polycarboxylic acid selected from the class consisting ofterephthalic acid, phthalic acid, 1,2,3-tricanboxybenzene,l,2,4-t-ricarboxybenzene, 1,3,5- tricarboxybenzene and1,2,4,5-tetracarboxybenzene, and from about 10 to about 250 parts of asurface active agent of the class consisting of dodecylbenzene sodiumsulfonate, mixed acids derived from tallow condensed with ethyleneoxide, and mixed alcohols derived from tallow condensed with ethyleneoxide.

References Cited by the Examiner UNITED STATES PATENTS 2,7 34,482 2/1956Seltzer 119-1 2,855,340 10/1958 Kent 167-53 2,890,980 6/ 1959 H'otchkiss167-53 2,907,693 10/1959 Price et a1 16765 2,994,639 8/1961 Carper et al167'65 3,011,943 12/1961 Rogers l6753 3,074,846 1/1963 Nichols 167-53LEWIS GOTTS, Primary Examiner.

SHEP K. ROSE, Assistant Examiner.

1. A METHOD OF AGUMENTING ANIMAL BLOOD CONCENTRATION OF ANTIBIOTICSSELECTED FROM THE CLASS CONSISTING OF 5-OXYTETRACYCLINE AND7-CHLOROTETRACYCLINE WHICH CONSISTS ESSENTIALLY IN FEEDING BIRDS ABOUT75 MG. PER KG. BODY WEIGHT OF AN ANTIBIOTIC SELECTED FROMTHE CLASSCONSISTING OF 5-OXYTETRACYCLINE AND 7-CHLOROTETRACYCLINE IN COMBINATIONWITH ABOUT 400 MG. PER KG. BODY WEIGHT OF A BENZENE POLYCARBOXYLIC ACIDOF THE CLASS CONSISTING OF TEREPTHALIC ACID, PHTHALIC ACID,1,2,3-TRICARBOXYBENZENE, 1,2,4-TRICARBOXYBENZENE,1,3,5-TRICARBOXYBENZENE, AND 1,2,4,5-TETRACARBOXYBENZENE, AND ABOUT 400MG. PER KG. BODY WEIGHT OF SURFACE ACTIVE AGENTS SELECTED FROM THE CLASSCONSISTING OF DODECYLBENZENE SODIUM SULFONATE, MIXED ACIDS DERIVED FROMTALLOW CONDENSED WITH ETHYLENE OXIDE AND MIXED ALCOHOLS DERIVED FROMTALLOW CONDENSED WITH ETHYLENE OXIDE.